Microbial Diversity 1999

On the hunt for mesophilic Korarchaeota

Jakob Zopfi, Max Planck Institute for marine Microbiology Celsiusstr.1, D-28359 Bremen, Germany

Introduction

In 1994 Barns et al. obtained two unusual archaeal clones from Obsidian Pool (Yellowstone) which did not fit into either the Euryarcimeota or the Crenarchaeota. After careful analysis of the data a new kingdom within the Archaea was proposed, the Korarchaeota (Barns et al., 1996). According to their data the Korarchaeota branch very near the root of the archaeal tree and the possibility exists that representatives of this new kingdom may display novel biological properties of relevance to our understanding of ancient organisms. Although not in pure culture yet a Korarchaeote has been reported to be a prominent species in an anaerobic, thermophilic continuos culture (Burggraf et al., 1997). In 1999 (Takai and Sako) presented data on 3 additional clones which grouped with the clones described by Barns et al. (1996). Furthermore, another clone that did not fit into one of the three kingdoms was described. The new korarchaeal clones were isolated from a shallow water hot vent site and from an acidic hot spring. If the Korarchaeota indeed represent a new kingdom within the Archaea, one might expect a greater phylogenetic and physiological diversity than described so far. One could speculate that more than thermophilic and acidophilic members of this kingdom exist. However, so far nobody has looked for Korarchaeota in samples from mesophilic environments. The scientific goal of this study is to expand the very limited phylogenetic diversity within the newly proposed kingdom of the Korarchaeota. The analysis of further clones from this lineage will help to clarifr the position of the Korarchaeota within the phylogenetic tree of life and might extend the range of habitats in which this almost untouched group of Archaea can be found Microbial Diversity 1999

Material and Methods

Genomic DNA was isolated from cyanobacterial mats from the Sippewissett salt marsh (Falmouth, Cape Cod) and from Guerrero Negro (Baja California, Mexico). Additionally, DNA from a hydrothermal vent site (Guaymas Basin, Mexico) was extracted. DNA was extracted from different depth within the core (5, 10, 15 cm.) which corresponds to in situ temperatures of 58, 90 and 100°C respectively. DNA was extracted using a Soil DNA KitTM (MO BlO, 12800-100-S). In case of the Guaymas sediments this method was not appropriate to isolate DNA and instead a modified phenol-chloroform-isopropylalcohol procedure was applied. To about 0.5 g (ww) of sample 895.tl of lysis buffer, 62.5 j.tlSDS (20%) and 0.5 g beads (0.2 mm, Biospec) were added. The whole sample was then “bead beaten” at full speed for 15 s (which might have been somewhat short). Then 42 j.tlProteinase K (20 mg/mi) was added to the mixture and incubated at 55°C for one hour. This step can be done in a rotating hybridization oven. The samples were divided into about 800 itl portions and then extracted with I00tI phenol-chloroform-isopropylalcohol (25:24:1) at 55°C for 10 mm. After centrifugation (10 mm., 14 krpm) the aqueous supematant was carefully removed and the extraction step was repeated two more times. The aqueous phases were pooled and DNA was precipitated by adding 0.1 vol. NaAc (3M, pH 5.2) and 2 vol. ethanol 20(H free, -20°C). The samples were kept overnight at -20°C and then spun down (14000 g, 20 mm., 4°C). The pellet was washed with 200j.tlice-cooled ethanol (70%) and spun down again (ca. 5 min.). The supernatant was withdrawn and the pellet was air dried and subsequently taken up in pure and sterile filtered water. In our case the DNA was dissolved in only 15j.tlof water.

PCR amplification The oligonucleotide sequences used for Korarchaeal rDNA amplification were as follows (Brunk and Eis, 1998): 236F, 5’-GAG GCC CCA GGRTGG GAC CG-3’ 626F, 5’-GTT AAA TCC GCC TGA AGA CA-3’ Either an universal- (U1391R) or an archaeal- (ARCH915R) specific sequence was used as the reverse primer. Arch2lF and Eub8F were used as archaeal and eubacterial specific forward primers. MicrobialDiversity1999

DNA was amplified on a Eppendorf Mastercycler Gradient using AmpliTaq TMGold polymerase (Perkin Elmer) and the following reaction mixture:

lOx Ampli Taq Buffer II 2.5 p.1

25 mM MgC12 2.5 p.1 dNTP’s 1 p.1

Primer forward 1 p.1

Primer reverse 1 p.1

1%BSA 1.25 p.1 dH2O 10.55 p.1

Ampli Taq Polymerase 0.2 p.1 Template DNA 5 p.!

Total volume 25 p.1

The quality (size) of PCR products was routinely checked by agarose-gel electrophoresis (1.25 % SeaKem w/v in O.5xTBE). PCR products were then cloned using the Invitrogen TOPO TA cloning kit, according to the descriptions of the kit. The transformed E. coli TOP10 cells were grown overnight on LB-Amp plates (50 p.g/mlampicillin, x-gal). Only white colonies were picked for a PCR of the insert.

RFLP digest (Restriction Fragment Length Polymorphism) In order to determine the diversity of the obtained PCR products of inserts an aliquot of the product was digested by means of restriction enzymes.

PCR product 10 p.1 2OdH 8 p.1 lOx Buffer2 2 p.1

Hind P1 0.1 p.1

MspI 0.05 p.1 The whole assay was incubated at room temperature for 1-3 hours (or overnight). Products were subsequently separated by gel electrophoresis (2% MetaPhor, 70 V). Only clones that exhibited a different RFLP pattern were sent for sequencing. Some of the sequences were determined in house on a PE ABI 310 capillary sequencer. The phylogenetic analysis of sequences was done with ARB (TU Munich) and BLAST MicrobialDiversity1999 searches were done on the web site of the Center for Biotechnology Information (www.ncbi.nlm.nih.gov/blast).

Results and Discussion

Bacterial mats: DNA was successfully isolated from the Sippewissett bacterial mat and the cynobacterial mat from Guerero Negro (Baja California, Mexico) using a soil DNA extraction kit. Without further purification but a 1/10 dilution step this DNA could be used for PCR. In order to determine the optimal annealing temperature and primer combinations for Korarchaeota 16S rDNA amplification a gradient PCR (52-72°C) with Baja cyanobacterial mat DNA and different sets of primer combinations was done (Fig. 1). Although the primers bound more specifically at higher temperatures 0(t = 64-66°C) there were still several other bands present which indicates unspecific binding of the primers to genomic DNA. Nevertheless, the appearance of PCR products of approximately the right size class may indicate the presence of Korarchaeota 16S rDNA. Attempts to purify the PCR products on a low melt agarose gel were partially successful but the cloning of the DNA bands that were cut out from the gel failed. Only very few colonies of transformed E. coli grew on LB-AMP-X-gal plates and most of them were blue. This indicates (I) that the transformation itself was not very efficient and (II) that almost all of the transformants contained a plasmid without insert. Eventually this was due to loss of A-tags at the ends of the PCR product which greatly diminishes the ligation efficiency.

Hydrothermal sediment: In contrast to the cyanobacterial mat samples the extraction of DNA from deep-sea vent sediments failed with the MO BlO Kit. A more elaborate procedure that involved phenol-chioroform-isoamylalcohol extraction steps was used to obtain DNA from these sediments. Even with this method, initially no DNA was detected in the extract by means of gel electrophoresis. Only after concentrating the extract about 15 times the DNA was concentrated enough for PCR. The low extraction efficiency could either be due to the high content of hydrocarbons present at that particular site or due to very low bacterial biomass. Evidence for the latter is given in Fig. 2 and 3. The photomicrographs of DAPI stained sediment samples show very little bacterial cells. FISH (fluorescence in situ hybridization) with an Archaea specific oligonucleotide probe indicate again the presence of only a few cells and most of the probe seem to bind unspecifically to inorganic sediment particles. Fig.

cation Cyanobacterial

1:

K624F/U1391R

with

PCR

different

products

mat

Baja

primer

of

K236F/U1391R

a

66°C

California

temperature

sets

specific

K236F/ARCH915R

(Mexico).

gradient

for

Korarchaeota.

66°C

(52-72°C)

64°C

amplifi

Sample: Fig. 2: Photomicrograph of a DAPI stained sediment sample from the Guaymas Basin (Mexico). The in situ temperature of this sediment layer was about 100°C. I 4 4.

I

Arch DAlI

Fig. 3: Photomicrographsof a Guaymas vent sediment sample. Top: phase contrast Bottom left: FISH with rhodamine labeled Archaea specific oligonucleotide probe. Bottom right: DAPI stained sample. Scale bar is valid for all three pictures. Microbial Diversity 1999

Eventually, a longer bead beating step (> 15s) at the beginning of the extraction procedure may have increased the DNA recovery. However, even the little DNA we had obtained was enough for some PCR reactions. Instead of starting directly with Korarchaeota specific primers this time the first amplification of the DNA was done with Eubacteria and Archae specific primers (Fig. 4). The eventually obtained archaeal PCR product would then be screened for Kor-sequences using the specific primers and amplification conditions described above. Surprisingly, neither in the Guaymas nor in the cyanobacterial mat samples from Guerrero Negro were archaeal PCR products of the expected size observed (Fig. 4.). The only specific amplification products were obtained with eubacterial primers in Guaymas sediments. However, the in situ temperature of these sediment samples was between 58 and 100°C at 15 cm depth, which is above the temperature range reported for Eubacteria. Thermophilic Eubacteria are often gram negative and show optimal growth between 40 and about 80 °C. So far only members of the genus Thermotoga and Aquifex are known to thrive at temperatures above 90°C, but not at 100°C. The so called hyperthermophillic microorganisms growing at 100°C and above are exclusively Archaea. A possible explanation for the lack of archaeal sequences could be that the beat beating step of only 15 s in the DNA extraction procedure preferentially destroyed eubacterial cells and left the more resistant Archaea unaffected. This, however, is an assumption and not based on real data. On the other side, eubacterial contaminants (introduced during sampling, sample treatment, DNA extraction...) could lead to an eubacterial signal although there were no Eubacteria present in the original sample, or there was even no DNA at all. In order to clarify these questions the PCR products were cloned in E. coli. After picking about 80 colonies 13 different (indicated by RFLP) clones were isolated and sent for sequencing. Unfortunately, only three of them could be sequenced. A BLAST search with three of the obtained sequences revealed Thermus and Actinothermus but also Nitrosococcus, Caulobacter and Propionigenium to be closest relatives. However the similarity between the clone sequence and the database hint was usually rather low (about 91%). It has to be stressed that the obtained sequences were short (about 150 nt) and of a rather poor quality, which makes a further interpretation of the data difficult. However, the fact that often thermophilic Eubacteria appeared in the BLAST search suggests that the obtained clones were indeed members of the original bacterial community and not simply contaminants. 1kB EubCont GB Eub GB Arch MB Eub MB Arch 1kB

Fig. 4: PCR products of Guaymas (GB) sediments and Baja mat (BM) with Eubacterial and Archaeal primers. Numbers indicate sample depth in cores (in cm). No

unspecifically confirmed different of Guaymas search also diversity Acad. Acad. Barns, diversity, Barns, terrestrial Doolitle, Takai, Burggraf,

8799. Visscher cyanobacterial suggestions Special

up

mesophilic

Nitrosococcus,

to

Sci. Sci.

with

K.,

S.M.,

S.M.,C.F.

thanks

100°C

Kor-specific

detected

W.F.

are

hot

thermophily

hot

S.,

Y. by

USA, USA,

three

and

R.E.

gratefully

P.

vent

DAPI Sako

water

to

(1996),

to and

mat a

Heyder,

practical

variety

sediment 91:

93:

Delwiche,

of

Scott

Fundyga,

in

site

thermophilic

(1999)

samples.

Caulobacter

environments. the

staining

a

1609-1913. 19188-9193.

forward

Yellowstone

was

and

At

acknowledged

Dawson,

obtained

of

N.

help

the

A particles

monophyly

extremely

13

Eis

M.W. J.D.

and

molecular

core

different

primers.

with

(1997),

korarchaeal and

Palmer,

Joel FISH.

sequences

Jeffries,

of

FEMS

Acknowledgments

than National

all

Propionigenium

Archaea.

Klappenbach

low.

Conclusions

for

the

from References

A

The

view eubacterial

DAPI

to

N.R.

pivotal

providing

MicrobioL

little

This

N.R.

microorganisms.

revealed

cell

environmental

Park

PCR

of

as

Pace

Proc.

problems.

archaeal was

and

Pace

well

Archaea

hot

products

clones

and

(1996).

Guaymas indicated DNA

Natl.

Thermus

Ecol.,

to

(1994). as

spring

Tom

be

FISH

diversity

was

group. Andreas Acad.

content

closest

rRNA

were

28:

Perspectives

Despite

environment.

Schmidt

Remarkable

by

sediment and bound

obtained.

177-188.

Sci.

obtained

the Nature,

in

sequences.

in

Actinothermus relatives. Microbial

Teske

marine

in low

sediment

USA,

rather

for

situ

and

A

on

DNA

385:

their

and

using archaeal

Proc.

BLAST

93,

temperatures

archaeal

Baja

and Diversity

Proc.

Piet

780.

useful from

yield

8797-

the

Nati.

but

Natl.

the

and 1999